1. Field of the Invention
The present invention provides salt crystals of non-natural stereoisomer forms of monatin and to the use thereof. More particularly, the present invention relates to salt crystals of non-natural stereoisomers of naturally occurring monatin (a (2S,4S) substance), which has excellent sweetening properties.
2. Discussion of the Background
As a result of modern eating habits, obesity arising from excessive ingestion of sugars and various diseases accompanied thereby have become a problem of medical and social importance. Accordingly, there has been a strong demand for the development of a low-calorie sweetener to replace sugar. In addition to the intensity of sweet taste, the sweetener demanded is requested to have many characteristics and essential features such as low calorie, safety, stability to heat and acid, quality of sweet taste, cost, etc.
At present, various kinds of sweeteners are used or proposed. For example, aspartame has been widely used as a sweetener due to its strong intensity of sweet taste (degree of sweet taste) and ease of mass-production. Of equal importance, aspartame also a proven safety record. Studies for derivatives of aspartame have been intensively performed.
In addition to the aspartame derivatives, sweet taste substances having various characteristics have been proposed as sweeteners and investigation for practical use has been conducted. Further, thaumatin, glycyrrhizin, stevioside, etc. derived from plants that are present in nature and able to be collected in large quantities are now used as natural sweeteners. Under such circumstances, there has been a demand for developing a sweet taste substance for practical use as a sweetener and having a strong degree of sweet taste.
Monatin is a naturally occurring amino acid derivative isolated from the bark of the roots of Schlerochiton ilicifolius, which is a plant naturally grown in the area of the north western Transvaal of South Africa. The structure of monatin was reported to be (2S,4S)-2-amino-4-carboxy-4-hydroxy-5-(3-indolyl)-pentanoic acid ((2S,4S)-4-hydroxy-4-(3-indolylmethyl)-glutamic acid; refer to the structural formula (1)) by R. Vleggaar, et al. (cf. R. Vleggaar, et al., J. Chem. Soc. Perkin Trans., 3095-3098 (1992)).
According to Vleggaar, et al, intensity of the sweet taste of a (2S,4S) substance (natural-type monatin) derived from the natural plant is reported to be 800- to 1400-fold greater than that of sucrose. Although various methods have been reported as a synthetic method for producing monatin, many of these relate to synthetic methods for a mixture of stereoisomers and there has been nearly no report where each of four stereoisomers having the same chemical structural formulae as the natural-type monatin is synthesized and isolated as a pure substance and properties thereof are investigated in detail. (With regard to examples of synthesis thereof, P. J. van Wyk, et al., ZA 87/4288, ZA 88/4220; Holzapfel, et al., Synthetic Communications, 24(22), 3197-3211 (1994); E. Abushanab, et al., U.S. Pat. No. 5,994,559 (1999); K. Nakamura, et al., Organic Letters, 2, 2967-2970 (2000), etc. may be referred to.)
P. J. van Wyk, et al (GB 2 205 834)) first disclosed monatin. Although the inventors determined by means of an X-ray crystal structure analysis that a stereoisomer present in nature and having a strong sweet taste is a (2S,4S) substance or a (2R,4R) substance, they reported that, by combining with the result of synthesis of a mixture of a (2S,4S) substance and a (2S,4R) substance from (2S)-aspartic acid, there is a high probability that a stereoisomer present in nature and having a strong sweet taste is a (2S,4S) substance. Then, in accordance with the above-mentioned document by R. Vleggaar, et al., a stereoisomer (steric structure) of monatin is reported as being present in natural plant is a (2S,4S) substance, only, and that its intensity of sweet taste is 800- to 1400-fold of sucrose. On the basis of this information, it is proper to conclude that a stereoisomer (steric structure) of monatin which is present in nature and is a substantial body of a strong sweet taste is a (2S,4S) substance.
In K. Nakamura, et al., they isolated hydrochlorides of a (2S,4S) monatin substance and of a (2S,4R) monatin substance. They reported that, with regard to the intensity of sweet taste thereof, the synthetic (2S,4S) monatin substance exhibited a sweet potency equivalent to that of the a natural specimen of monatin (a (2S,4S) substance) and the synthetic (2S,4R) monatin substance exhibited a slightly sweet taste presumably due to monatin (a (2S,4S) substance), which is thought to be present as an impurity, although the specific intensity of the sweet taste was not reported. Thus, although this document is the first example referring to the intensity of the sweet taste of a non-natural-type stereoisomer (other than a (2S,4S) substance) of monatin, it is reported that hydrochloride of a (2S,4R) monatin substance has almost no sweet taste.
On the other hand, T. Kitahara, et al. report a selective synthetic method for each stereoisomer of monatin as a sweetener (sweetening agent) but do not report the degree of sweet taste of each stereoisomer (refer to T. Kitahara, et al., Abstracts of Papers Presented at the General Meeting in 2000 of the Agricultural Chemical Society of Japan, 3B128β (page 221)).
When the information as mentioned above is taken into consideration as a whole, the followings have been noted.                (1) A stereoisomer (steric structure) of monatin that is present in nature and has an intensity of sweet taste of as strong as 800- to 1400-fold is a (2S,4S) substance; and        (2) With regard to other non-natural-type stereoisomers of monatin, although there are examples where they are isolated in small quantities, there is no example where the pure substance is isolated, purified, and the intensity of the sweetness assessed.        
Thus, heretofore, there have been no clear data for the intensity of sweet taste of each stereoisomer of monatin in practical concentrations corresponding to 5 to 10% sucrose concentrations except the monatin form that is obtained from nature (a (2S,4S) substance). Accordingly, it is not possible to know from the prior art whether non-natural stereoisomers of monatin except monatin (a (2S,4S) substance) are useful as a sweetener. In other words, reliable information for the degree of sweet taste (intensity of sweet taste) except monatin derived from nature (a (2S,4S) substance) is little and, when the prior art is considered as a whole, the present situation is nothing but to conclude that non-natural-type monatin stereoisomers except monatin (a (2S,4S) substance) have low degree of sweet taste and are unable to be expected for their utility as sweeteners.
One of the causes therefor is that, until now, there has been found no method whereby each of the above-mentioned stereoisomers may be synthesized, isolated and purified.
Accordingly, there exists a demand for at least several hundred milligrams or more of the naturally occurring monatin, as well as the three non-natural stereoisomers thereof. More specifically, there exists a demand for isolated and purified forms of the same for assessment of the optical purity and intensity of sweetness thereof. Thereby the utility of monatin (a (2S,4S) substance) and stereoisomers thereof (i.e., non-natural-type stereoisomers) as sweeteners may be assessed and ultimately a sweetener containing a component having a high utility may be developed.
With regard to a method of separation of each stereoisomer in a high purity, there will be firstly a crystallizing method. Therefore, some explanations will be made for crystals of monatin (including the forms of free compounds, salts, etc.) as follows.
The state of the prior art is summarized as follows. R. Vleggaar, et al. report that crystals of a free compound of monatin (a (2S,4S) substance) are prepared from a mixed solvent of water, acetic acid and ethanol (1:1:5) and its melting point is described as 216-220° C. P. J. van Wyk, et al., disclosed a melting point of a free compound (crystalline solid) of monatin (a (2S,4S) substance) is described as 247-265° C. (decomposition) while, with regard to various salts, they are reported to be amorphous solids. In C. W. Holzapfel, et al., crystals of a free compound of a mixture of a (2S,4S) substance and a (2R,4R) substance of synthetic monatin are prepared by crystallization for two times from a mixed solvent of water and acetic acid (10:1) and the melting point is reported to be 212-214° C.
Accordingly, with regard to non-natural stereoisomers of monatin and a mixture of such a plurality of stereoisomers except the above two examples such as a free compound of a (2S,4S) monatin substance, not only free compound but also various salts have not been isolated in a crystalline state whereby their physical property data and other information have not been known at all.
Thus, a crystallizing method does not exist at present. Such a method would be the simplest and most effective method for purification as compared with conventional purifying methods such as ion-exchange chromatography and also for crystals prepared thereby except the two cases of a free compound of monatin (a (2S,4S) substance) and a mixture of free compounds of (2S,4S) substance and (2R,4R) monatin substance. At present no knowledge exists at all for crystals of salts. Accordingly, in view of a practical application of various stereoisomers of monatin as sweeteners, there exists a critical demand for these salt forms.